Cereal grain is one of the most important renewable energy sources for humans and animals. Since over 90% of corn grain is used for animal feed, corn is one of the most important crops for animal nutrition. Grain of non-specialty yellow dent corn consists of 60-70% starch, 8-10% protein, and 3-4% oil. However, despite these valuable feed components, non-specialty yellow dent corn does not contain sufficient calories and essential amino acids to support optimal growth and development in most animals. Therefore, to compensate for these shortcomings, it is necessary to supplement yellow dent corn-based feed. Most commonly, yellow dent corn is supplemented with soybean meal and amino acids to improve the amino acid composition and caloric density of the feed. Unfortunately, animals lack the enzymes necessary to digest the non-starch based polysaccharides present in soybean meal, and corn/soybean feed mixtures result in high manure volume. In addition, soybean meal is expensive. Furthermore, to improve caloric content, corn-based animal feed is also supplemented with fats, such as animal offal and feed-grade animal and vegetable fats, which may include by-products of the restaurant, soap, and refinery industries. Use of animal offal to supplement cattle feed has been discontinued because of its association with bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Improvements to the nutritional qualities of corn grain will increase feed efficiency and reduce environmental impact and other costs associated with meat production.
The developing cereal grain seed is a remarkable factory, composed of pericarp, embryo, and endosperm. Most oil is synthesized and stored in the embryo, while the endosperm tissue contains the starch-based energy store. Protein is stored both in the embryo and endosperm. In all cereal crop plants, sucrose is delivered to the seed from the leaves and converted into hexose sugars, such as glucose, which in combination with amino acids is used for metabolism and synthesis of storage compounds such as starch, protein, and oil.
Since starch comprises 70% of cereal grain dry weight, starch biosynthesis plays an important role in determining seed yield and quality. The committed steps of starch biosynthesis involve at least three enzymatic reactions. First, adenosine diphosphate glucose (ADPG) is synthesized from glucose 1-phosphate and adenosine triphosphate (ATP), a reaction which is catalyzed by ADP-glucose pyrophosphorylase (AGPase). Then, starch synthase elongates α-1,4 glucan chains by transferring glucose from ADPG to an acceptor chain. Finally, branching enzyme hydrolyzes an elongated α-1,4 glucan chain and simultaneously transfers it to an acceptor chain to form an α-1,6 linkage.
In cereal grain seed endosperm tissue cells, the conversion of glucose to ADPG by AGPase occurs primarily in the cytosol, and ADPG is imported into the amyloplast by a specific ADPG transporter protein. There is minor AGPase activity in the endosperm amyloplast compartment (hereinafter referred to as the plastid or plastid compartment) of monocots. In corn endosperm, for example, the plastidial AGPase activity represents less than ten percent of the overall AGPase activity of the seed. In contrast, in dicot crops AGPase is localized primarily (and possibly exclusively) in the plastid, synthesizing ADPG primarily in the plastid compartment of the cell. Since AGPase is encoded by a nuclear gene, localization of AGPase in the plastid requires that the protein be first expressed in the cytosol and then translocated into the plastid by a plastid transit peptide. If the plastid transit peptide is lacking, the plant's AGPase activity will remain in the cytosol.
AGPase is considered one of the limiting steps in starch biosynthesis in plants and glycogen synthesis in bacteria. Much effort has been focused on improving seed quality by genetic engineering of AGPase to change starch content of cereal grain seeds.
U.S. Pat. No. 5,792,920 discloses isolation of genes encoding AGPases from wheat endosperm and leaf. U.S. Pat. No. 5,792,920 further discloses incorporation of the disclosed genes into a cereal plant genome in the sense or antisense direction, to improve or reduce the plant's ability to synthesize starch.
U.S. Pat. Nos. 5,498,831 and 5,773,693 disclose cDNAs encoding the large subunit (SH2) and small subunit (BT2) of pea AGPase. U.S. Pat. Nos. 5,498,831 and 5,773,693 also disclose that overexpression of these genes requires a plastid transit sequence for proper sub-localization of the heterotetrameric enzyme.
U.S. Pat. No. 6,232,529 discloses a method for enhancing accumulation of oil beyond normal levels in the embryo of a corn seed, by reducing starch production by diminishing or abolishing the activity of AGPase in the embryo, but not in other tissue.
U.S. Pat. No. 6,486,383 discloses transgenic potato plants containing either the wheat brittle 2 gene (the small subunit of wheat AGPase) or the wheat shrunken-2 gene (the large subunit of wheat AGPase). The transgenic potato plants of U.S. Pat. No. 6,486,383 demonstrated increased AGPase activity.
WO 93/09237 discloses a method of improving the starch and sugar content to sweet corn by manipulating the timing of expression of the sh-2 (large) and bt-2 (small) subunits of the heterotetrameric maize AGPase.
WO 94/24292 discloses DNA sequences of the large and small subunits of the barley endosperm AGPase, including targeting expression of either or both subunits to plant endosperm tissue using DNA sequences encoding transit peptides.
WO 98/10082 discloses a mutant of the maize Sh2 (AGPase large subunit) gene designated Sh2-m1Rev6, which results in increased seed weight, but which demonstrates no higher percentage of starch as compared to other Sh2 alleles.
WO 98/22601 and WO 99/58698 disclose mutants of the maize endosperm large subunit gene, which are purported to improve yield when present in plants grown under heat stress.
WO 99/07841 discloses up-regulated allosteric mutants of plant AGPases, which result in increased starch production, increased yield, increased plant size, increased growth rate, and increased numbers of seeds when transformed into Arabidopsis. 
U.S. Pat. Nos. 5,349,123; 5,969,214; and 6,538,181 disclose DNA sequences encoding bacterial glycogen biosynthetic enzymes, including a glgC mutant derived from E. coli 618 designated pGlgC-37. U.S. Pat. Nos. 5,349,123; 5,969,214; and 6,538,181 state that the AGPase from E. coli 618 differs from that of E. coli K12 at five amino acids, and that the translated amino acid sequences of pGlgC-37 differs from the AGPase of E. coli 618 only at position 361, by a substitution of aspartate for asparagine. U.S. Pat. Nos. 5,349,123; 5,969,214; and 6,538,181 also disclose joining the bacterial glycogen synthetic enzyme genes with a sequence encoding a transit peptide that provides for translocation of the enzyme to a plastid of a plant. These patents predicted that expressing bacterial glycogen biosynthetic enzymes in plant plastids would result in modulation of the starch content of the plant.
The AGPase from E. coli 618 has been extensively studied. Lee, et al. (1987) Nucleic Acids Res. 15, 10603 also discloses that the AGPase from strain 618 differs from that of E. coli K12 strain 3000 (commonly designated as the wild type enzyme) at five amino acid residues, valine to alanine at position 161, valine to alanine at position 166, threonine to isoleucine at position 189, lysine to glutamate at position 296, and glycine to aspartate at position 336. However, Kumar, et al. (1989) J. Biol. Chem. 264, 10464-10471 discloses that the substitutions at positions 161, 166, and 189 previously reported for the 618 AGPase were sequencing errors, and that the 618 enzyme did not differ from the wild type AGPase at these three positions. Furthermore, Meyer, et al. (1993) reported that the sequence of the wild type enzyme at position 296 is glutamate and not lysine as previously reported, and thus that the 618 AGPase differs from the wild type enzyme only by the glycine to aspartate substitution at position 336.
U.S. Pat. No. 6,538,178, related U.S. Pat. Nos. 6,538,179; 5,498,830 and 5,608,149, WO 91/19806, and EP 634491 disclose that transformation into plants of wild type and mutant E. coli AGPase genes, including the mutant gene from strain 618 (designated therein as glgC16), in fusion with a plastid targeting transit peptide, results in increased starch content. U.S. Pat. No. 6,538,178 specifically exemplifes increases in starch content of transformed tobacco callus, potatoes, and tomato. U.S. Pat. Nos. 5,498,830 and 6,538,179, continuations-in-part of the parent application of U.S. Pat. No. 6,538,178, disclose additional transformations of a plastid transit peptide-glgC16 gene fusion into potato, tomato, canola, and maize. U.S. Pat. Nos. 5,498,830 and 6,538,179 disclose that canola seeds transformed with a plastid transit peptide-glgC16 gene fusion demonstrated increased starch content and decreased oil content, while protein content and moisture were not significantly changed. U.S. Pat. Nos. 5,498,830 and 6,538,179 further disclose that maize Black Mexican Sweet callus transformed with a plastid transit peptide-glgC16 gene fusion demonstrated two to threefold increases in starch levels, as compared with control lines.
Sakulsingharoj, et al (2004) Plant Science 167, 1323-1333 discloses rice transformed with an E. coli AGPase triple mutant (described therein as R67K, P295D, G366D), which displays up to 90% of the catalytic activity of the fully activated wild-type enzyme in the absence of any activators. The triple mutant AGPase gene was transformed both with and without a transit peptide sequence, under control of the rice glutelin Gt1 promoter. Rice transformants thus generated expressed the bacterial enzyme either in the amyloplast or in the cytoplasm of developing seeds. The authors noted a positive correlation between increases in cytoplasmic AGPase activity, 14C-sucrose incorporation into starch, and seed weight. However, rice plants exhibiting elevated AGPase activity in amyloplast exhibited variable responses.
U.S. Pat. No. 6,483,011 discloses methods for generating AGPase mutants, which increase starch formation, or which increase the accumulation or depletion of certain starches. U.S. Pat. No. 6,483,011 discloses a mutant E. coli strain, which lacks AGPase activity and carries an unknown mutation in the E. coli glgC gene designated glgC3. The glgC3 disclosed in U.S. Pat. No. 6,483,011 is a loss of function mutant.
WO 98/44780 discloses the DNA and protein sequences of a mutant AGPase from E. coli designated glgC3, which contains two mutations, a proline to aspartic acid substitution at amino acid 295 and a glutamic acid to lysine substitution at amino acid 296. Rice transformants containing the glgC3 gene are also disclosed in WO 98/44780.
Bacterial AGPase is a homotetramer encoded by the glgC gene, while plant AGPase is a heterotetramer. Based on specificity for activator and inhibitor, AGPases have been grouped into classes 1 through VII, as summarized in Ballicora et al. (2003) Microbio. Mol. Biol. Rev. 67, 213-225, Table 1. With some exceptions, prokaryotic AGPases are activated by fructose-1,6-bis-phosphate (FBP), and inhibited by adenosine monophosphate (AMP), and higher plant AGPases are activated by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate.
U.S. Pat. Nos. 4,885,357; 4,886,878; 5,003,045; 5,576,203; 5,487,991; 5,589,615; 5,623,067; 5,258,300; 5,082,993; 5,589,616; 5,215,912; 5,367,110; 5,559,223; 5,451,516; 5,633,436; 5,530,192; 5,545,545; and 5,633,436 disclose various methods for genetic engineering of plants to increase the content of one or more amino acids. Since the amino acid content of seed is primarily (90-99%) determined by the bound amino acid (protein composition) and to a much less extent (1-10%) by the free amino acid pool, there are serious challenges to approaches which result in increases of free amino acids to improve nutritional value. One challenge is that increased free amino acid concentration is not always associated with an increase in total amino acid, since the free amino acid content is such a small percentage of the total amino acid and the flux and incorporation of free amino acid into protein may become limiting. Secondly, due to accumulation of free amino acids or reduced amino acid catabolism, amino acid changes in these studies are often associated with adverse agronomic performance, such as stunted growth, therefore affecting marketability. Third, the targeted approach often leads to changes in one or two amino acids, which may limit the market value of the grain so produced.
A need continues to exist for corn grain with increased levels of certain amino acids and oil, and which has desirable agronomic characteristics.